Fluorescent nucleic acid base analogs can be widely used in fluorescent labeling of nucleic acids. In the fluorescent labeling of nucleic acids, in general, a fluorescent dye is linked to any of natural bases, and this modified base is introduced into DNA or RNA by chemical synthesis or enzymatic reaction (replication or transcription). However, this method may inactivate the function of a nucleic acid by the fluorescent dye moiety, because it significantly protrudes from the structure of the nucleic acid and undergoes stacking with any base of the nucleic acid. In addition, the fluorescent dye is linked to a natural base; hence, such a base cannot be introduced into a specific position of a nucleic acid by replication or transcription. In contrast, the fluorescent nucleic acid base analog can label a nucleic acid while maintaining the structure and the function of the nucleic acid. Further, when the analog functions as an unnatural base pair in replication or transcription, it can be introduced at a specific position of DNA or RNA.
For example, 2-aminopurine and 2,6-diaminopurine are known as fluorescent nucleic acid base analogs (Patent Document 1 and Non-Patent Documents 1 to 4). The fluorescence intensities of these base analogs, however, are not so high. Further, when these base analogs are introduced into nucleic acids, the fluorescence of the analogs is quenched by stacking with neighboring bases. These base analogs are adenine (A) analogs and can be introduced into DNA or RNA by replication or transcription as complementary bases of tymine (T). However, the incorporation efficiencies of the adenine analogs are low, and they are introduced at positions corresponding to A of nucleic acids in replication or transcription and thus cannot be introduced at a specific position. If a nucleic acid has only one A, the base analog can be introduced at the position of the A; however, such a sequence of nucleic acid is a very particular case and thus lacks versatility. In addition, though these base analogs can substitute for A in DNA or RNA as analogs of A, substitution of such a base analog for another base (such as G, C, or T) may reduce the function of the nucleic acid.
The present inventors have intensively developed the third base pairs (unnatural base pairs) for expanding genetic information of DNA. The present inventors have successfully developed several unnatural base pairs that function in replication or transcription, such as an s-y base pair (s: 2-amino-6-thienylpurine, y: pyridin-2-one), a v-y base pair (v: 2-amino-6-thiazolyl purine), an s-Pa base pair (Pa: pyrrolo-2-carbaldehyde), a Ds-Pa base pair (Ds: 7-(2-thienyl)-imidazo[4,5-b]pyridine), and a Ds-Pn base pair (Pn: 2-nitropyrrole) (Non-Patent Documents 5 to 10). The unnatural bases s and v have fluorescence, and the inventors have also reported an analytical technique for local structure of nucleic acid using these unnatural bases. However, the fluorescence intensity of s is not so high, and s has a maximum excitation wavelength of 348 nm and a fluorescence wavelength of 435 nm; hence, a nucleic acid base analog having these wavelengths shifted to longer wavelengths is desired. Though v has a higher fluorescence intensity than s, it has low stability as a compound, which can be readily degraded under basic conditions. Its use is thus limited. With regard to the Ds-Pa base pair and the Ds-Pn base pair, DNA containing these unnatural base pairs can be amplified by PCR. Thus, these base pairs are very useful. However, the fluorescence of Ds by excitation at a wavelength of 350 nm or more is substantially invisible to the naked eye.
Accordingly, development of unnatural fluorescent bases that can be introduced into specific positions in DNA or RNA by replication or transcription will enable a novel method of fluorescent labeling of a nucleic acid to be established.
Incidentally, a base analog that forms a base pair with any natural base with substantially the same stability is called a universal base, and, for example, pyrrole-3-carboxamide, 3-nitropyrrole, and 5-nitroindole are known as such universal bases (Patent Documents 2 to 4 and Non-Patent Documents 11 to 17). However, there is a need for an unnatural base as a universal base having a higher thermal stability in the technical field of labeling a functional nucleic acid with an unnatural base.